Test strip for short-wave near infrared immunofluorescence chromatographic detection and use thereof

ABSTRACT

Provided are a test strip for short-wave near infrared immunofluorescence chromatographic detection, a system for immunofluorescence chromatographic detection and a method for quantifying an analyte in a sample. The test strip for short-wave near infrared immunofluorescence chromatographic detection includes: a body, defining a sample region, a binding region, a detecting region and an adsorbing region connected with one another sequentially; a first antibody, labeled with a fluorescent microsphere, coated on the binding region and configured to specifically recognize an analyte; a detecting line and a quality control line, located in the detecting region, wherein the detecting line closes to the binding region; a second antibody, coated on the detecting line and configured to specifically recognize the analyte; and a third antibody, coated on the quality control line and configured to specifically recognize the first antibody, in which the fluorescent microsphere is a near-infrared II polymer fluorescent microsphere.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priorities to and benefits of ChinesePatent Application Nos. 201710607567.9, 201710607934.5 and201710608436.2, all filed with the State Intellectual Property Office ofP. R. China on Jul. 24, 2017, the entire contents of which areincorporated herein by reference.

FIELD

The present disclosure relates to the field of biotechnology, inparticular, to a test strip for short-wave near infraredimmunofluorescence chromatographic detection, a system forimmunofluorescence chromatographic detection and a method forquantifying an analyte in a sample.

BACKGROUND

A polymer fluorescent microsphere, as a special function microsphere, iscapable of encapsulating tens of thousands to hundreds of thousands offluorescent molecules for one microsphere, such that labeling efficiencyand resistance to photobleaching of the fluorescent molecules are bothenhanced, thereby improving sensitivity of fluorescence detectiongreatly. Besides, the polymer fluorescent microsphere is allowed to bemodified from the outside with one or more functional groups (such as acarboxy group, an amino group and an aldehyde group) in a very flexiblyway, which benefits for covalent coupling to a protein (such as anantibody) and improvement of stability and labeling efficiency of anlabeling agent. At present, the polymer fluorescent microsphere has beenwidely used in labeling, tracing, detecting, imaging, enzymeimmobilizing, medical immunology, high-throughput drug screening and soon. However, the traditional polymer fluorescent microsphere emitslights merely in a visible region with an emitting wavelength below 780nm, resulting in poor penetrability and intense background fluorescencewhen applied in imaging of living organisms, cells or tissues,diagnosing in vitro, and so on. Meantime, since the traditional polymerfluorescent microsphere is generally with low quantum efficiency andrelative closer interval (around 20 nm) between an exciting wavelengthand an emitting wavelength, which results in poor analysis sensitivity,there exits high demand on a color filter for the fluorescencedetection.

Therefore, there is still a need to improve the polymer fluorescentmicrosphere.

SUMMARY

Embodiments of the present disclosure seek to solve at least one of theproblems existing in the related art to at least some extent.

An object of the present disclosure is to provide a test strip forshort-wave near infrared immunofluorescence chromatographic detection, asystem for immunofluorescence chromatographic detection and a method forquantifying an analyte in a sample. The test strip for short-wave nearinfrared immunofluorescence chromatographic detection proposed by thepresent disclosure includes an antibody labeled with a fluorescentmicrosphere which has advantageous characteristics of high quantumefficiency, an exciting wavelength less than 1000 nm, such as at 365 nmor 740 nm, and an emitting wavelength at 1000 nm to 1700 nm, and strongpenetrability and low background interference during fluorescencedetection, thus improving accuracy, sensitivity and precision ofdetected results.

In a first aspect, the present disclosure provides in embodiments a teststrip for short-wave near infrared immunofluorescence chromatographicdetection, including:

a body, defining a sample region, a binding region, a detecting regionand an adsorbing region connected with one another sequentially;

a first antibody, labeled with a fluorescent microsphere emitting awavelength in a range of 1000 nm to 1700 nm under an excitation lightless than 1000 nm, coated on the binding region and configured tospecifically recognize an analyte;

a testing line and a quality control line, located in the detectingregion, in which the testing line closes to the binding region;

a second antibody, coated on the testing line and configured tospecifically recognize the analyte; and

a third antibody, coated on the quality control line and configured tospecifically recognize the first antibody,

wherein the fluorescent microsphere is a near-infrared II polymerfluorescent microsphere prepared by the following steps:

1) dissolving a fluorochrome in a water-immiscible organic solvent, thusobtaining a fluorochrome solution;

2) distributing a polymer microsphere into a sodium dodecyl sulfonatesolution, thus obtaining a microsphere solution with the polymermicrosphere as a carrier for the fluorochrome;

3) subjecting a first mixture of the fluorochrome solution and themicrosphere solution to ultrasonic treatment, thus obtaining anemulsion;

4) swelling the emulsion such that the fluorochrome solution entersnanopores formed during swelling of the polymer microsphere, thusobtaining a second mixture; and

5) heating the second mixture to volatilize the organic solvent, suchthat the fluorochrome is crystallized out and encapsulated in thenanopores, thus obtaining the near-infrared II polymer fluorescentmicrosphere.

In some embodiments of the present disclosure, the fluorochrome isselected from the group consisting of organic molecules including thoseshown as formula (I), formula (II) or formula (III), or carbonnanotubes, or PbS, PbSe or InAs quantum dots, or rare earthnanoparticles.

In embodiments of the present disclosure, the formula (I) is4,8-(5-(9,9-di(6-bromohexyl)-9H-fluoren-2-yl)-2,3-dihydrothieno[3,4-b][1,4]dioxine)-1H,5H-benzo[1,2-c:4,5-c′]bis([1,2,5]thiadiazole);the formula (II) is4,8-(5-(9,9-dihexyl-9H-fluoren-2-yl)-2,3-dihydrothieno[3,4-b][1,4]dioxine)-1H,5H-benzo[1,2-c:4,5-c′]bis([1,2,5]thiadiazole);and the formula (III) is2,3-dihydrothieno[3,4-b][1,4]dioxine)-1H,5H-benzo[1,2-c:4,5-c′]bis([1,2,5]thiadiazole),thus improving accuracy, sensitivity and precision of the resultdetected by the present test strip

In some embodiments of the present disclosure, the analyte is cardiactroponin, the first antibody is an antibody I against cardiac troponin,the second antibody is an antibody II against cardiac troponin, and thethird antibody is a secondary antibody, preferably a goat-anti-mouseantibody, in which the first antibody recognizes the analyte at a firstsite different from a second site recognized by the second antibody,thus improving accuracy, sensitivity and precision of the resultdetected by the present test strip.

In some embodiments of the present disclosure, the fluorochrome in thefluorochrome solution has a concentration of 1 mg/mL to 50 mg/mL, sothat the polymer microsphere is capable of encapsulating morefluorochrome, thereby further improving sensitivity of fluorescencedetection.

In some embodiments of the present disclosure, the organic solvent is atleast one selected from the group consisting of ethyl acetate,dichloromethane, trichloromethane, 1,2-dichloroethane and aromatichydrocarbons, preferably dichloromethane.

In some embodiments of the present disclosure, the polymer microsphereis at least one selected from the group consisting of polystyrenemicrospheres, poly (methyl methacrylate) microspheres, polyformaldehydemicrospheres and poly (lactic acid-co-glycolic acid) microspheres whichcan be well distributed in an aqueous solution and allows to be modifiedat its surface with different radicals in a flexible way, therebyfacilitating to subsequent coupling. Therefore, the near-infrared IIpolymer fluorescent microsphere can be obtained effectively with strongpenetrability, low background interference and excellent dispersibilityin the aqueous solution.

In some embodiments of the present disclosure, the polymer microspherehas a particle size of 20 nm to 1000 nm, so that the polymer microsphereis capable of encapsulating more fluorochrome, thereby further improvingsensitivity of fluorescence detection.

In some embodiments of the present disclosure, in the step 2), thepolymer microsphere is distributed into the sodium dodecyl sulfonatesolution in a mass/volume ratio of 10 mg/mL to 200 mg/mL, thereby notonly guaranteeing the polymer microsphere to well disperse in the sodiumdodecyl sulfonate solution, but also allowing the polymer microsphere tobe fully in contact with dichloromethane during the subsequent swelling,such that the polymer microsphere can be swelled to a maximal extent.

In some embodiments of the present disclosure, the first mixtureincludes the fluorochrome solution and the microsphere solution in avolume ratio of 1:5 to 1:20, such that dichloromethane is in a properamount for swelling the polymer microspheres thoroughly, thus furtherincreasing encapsulation efficiency and enhancing weight of thenear-infrared II polymer fluorescent microsphere.

In some embodiments of the present disclosure, in the step 3), the firstmixture includes the fluorochrome and the polymer microsphere in a massratio of 0.1:100 to 30:100. Therefore, each microsphere is capable ofencapsulating tens of thousands to hundreds of thousands of fluorescentmolecules, thereby improving sensitivity of fluorescence detectiongreatly.

In some embodiments of the present disclosure, in the step 4), theemulsion is swelled at 10° C. to 50° C. under stirring for 1 hour to 10hours, such that the polymer microsphere can be swelled sufficiently inthe presence of dichloromethane, which ensures the fluorochrome enteringnanopores formed during swelling of the polymer microspheresuccessfully.

In some embodiments of the present disclosure, in the step 5), thesecond mixture is heated at a temperature of 50° C. to 90° C., such thatdichloromethane can be volatilized completely in a short time period.

In another aspect, the present disclosure provides in embodiments asystem for immunofluorescence chromatographic detection, including afluorescence immunity analyzer, and a test strip for short-wave nearinfrared immunofluorescence chromatographic detection described in thefirst aspect. As the present test strip can provide a fluorescencesignal which can be analyzed by the fluorescence immunity analyzer, ananalyte applied to the test strip can be detected qualitatively orquantitatively. Therefore, use of the system for the immunofluorescencechromatographic detection according to embodiments of the presentembodiments contributes to obtaining a detection result with highaccuracy, sensitivity and precision.

In still another aspect, the present disclosure provides in embodimentsa method for quantifying an analyte in a sample, including 1) applyingthe sample to a sampling region of a test strip for short-wave nearinfrared immunofluorescence chromatographic detection described in thefirst aspect; 2) determining a fluorescence signal generated in the teststrip for the immunofluorescence chromatographic detection; and 3)quantifying the analyte in the sample based on the fluorescence signaldetermined, thus contributing to obtaining a detection result with highaccuracy, sensitivity and precision, in a simple way.

In an embodiment of the present disclosure, the sample is serum.

In an embodiment of the present disclosure, the fluorescence signal isdetermined by a fluorescence immunity analyzer.

In an embodiment of the present disclosure, the fluorescence immunityanalyzer is equipped with a color filter at a wavelength of 800 nm, thusbenefiting for obtaining fluorescence with high intensity, andfacilitating to observing and quantifying the fluorescence obtained.

In an embodiment of the present disclosure, the method for quantifyingthe analyte in the sample further includes determining fluorescencesignals in both a testing line and a quality control line in the step2); and quantifying the analyte in the sample based on a ratio of thefluorescence signal generated in the testing line to the fluorescencesignal generated in the quality control line.

In an embodiment of the present disclosure, the method for quantifyingthe analyte in the sample further includes quantifying the analyte inthe sample by means of a standard curve, based on the ratio of thefluorescence signal generated in the testing line to the fluorescencesignal generated in the quality control line, in which the standardcurve is created with serial cardiac troponin standards inconcentrations of 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.2 ng/mL,2.0 ng/mL, 1.0 ng/mL, 0.5 ng/mL, 0.2 ng/mL and 0 ng/mL, thuscontributing to improving accuracy of a detected result.

DESCRIPTION OF DRAWINGS

The foregoing and/or additional aspects and advantages of the presentdisclosure will become apparent and be readily understood by combiningthe description of the embodiments with the drawings.

FIG. 1 is a structural representation showing a test strip forshort-wave near infrared immunofluorescence chromatographic detectionaccording to embodiments of the present disclosure.

FIG. 2 is a flow chart showing a method for preparing a near-infrared IIpolymer fluorescent microsphere according to embodiments of the presentdisclosure.

FIG. 3 is a flow chart showing a method for preparing a near-infrared IIpolymer fluorescent microsphere according to other embodiments of thepresent disclosure.

FIG. 4 shows absorption spectrums of and fluorescence spectrums emittedrespectively by fluorochrome represented by formula (I), (II) or (III)which is contained in near-infrared II polymer fluorescent microspheresaccording to embodiments of the present disclosure.

FIG. 5 shows scanning electron microscope photographs of carboxylicpolystyrene pellets and carboxylic polystyrene fluorescent microspheresaccording to embodiments of the present disclosure.

FIG. 6 are schematic graphs showing a carboxylic polystyrene microspheredispersion, a fluorochrome solution and a carboxylic polystyrenefluorescent microsphere solution respectively.

FIG. 7 shows fluorescent photographs and fluorescent spectrums ofcarboxylic polystyrene fluorescent microspheres under irradiation withan excitation light at a wavelength of 740 nm according to embodimentsof the present disclosure.

FIG. 8 is a structural representation showing a test strip forshort-wave near infrared immunofluorescence chromatographic detectionaccording to other embodiments of the present disclosure.

FIG. 9 is a structural representation showing a system forimmunofluorescence chromatographic detection according to embodiments ofthe present disclosure and a fluorescent spectrum of an analyte in asample detected by the system.

FIG. 10 are fluorescent photographs of cardiac troponin respectively atconcentrations of 0 ng/mL and 20 ng/mL detected by test strips forimmunofluorescence chromatographic detection according to embodiments ofthe present disclosure.

FIG. 11 are fluorescent spectrums of cardiac troponin at concentrationsfrom 0 ng/mL to 50 ng/mL in a testing line and in a quality control linerespectively according to an embodiment of the present disclosure.

FIG. 12 are fluorescent spectrums of cardiac troponin at concentrationsfrom 0 ng/mL to 80 ng/mL in a testing line and in a quality control linerespectively according to another embodiment of the present disclosure.

FIG. 13 is illustrative graphs showing standard curves of cardiactroponin according to embodiments of the present disclosure.

FIG. 14 is illustrative graphs showing comparison between resultsobtained by the present test strips for short-wave near infraredimmunofluorescence chromatographic detection according to embodiments ofthe present disclosure and results obtained from clinical detections.

DETAILED DESCRIPTION

The exemplary embodiments of the present disclosure are described indetail below, which are intended to be illustrative of the disclosureand are not to be construed to limit the disclosure.

It should be noted that the terms “first” and “second” are just intendedto be illustrative and are not to be construed as indicating oranticipating a relative importance or the number of technical featuresindicated. Thus, a feature that is defined as “first” or “second” mayexpressly or implicitly include one or more features. Further,throughout the description of the present disclosure, term “plural”includes two or more, unless otherwise specified.

The present disclosure is accomplished by the present inventors based onthe following discoveries.

Because of disadvantageous characteristics of poor penetrability andintense background fluorescence, low quantum efficiency and relativecloser interval between an exciting wavelength and an emittingwavelength, the existing method for fluorescence detection using thetraditional polymer fluorescent microsphere is in low sensitivity andwith high demand on a color filter used. The near-infrared fluorescentmicrosphere, especially the near-infrared II fluorescent microsphere hasadvantageous characteristics of strong penetrability and low backgroundinterference during the fluorescence detection, thereby having promisingprospects in terms of live imaging, biolabeling detection and so on.However, there still exist limitations on the existing method forpreparing the near-infrared II fluorescent microsphere, for example, ahydrophobic fluorochrome cannot be directly applied in live imagingbecause of strong hydrophobicity per se, and thus requiring hydrophilicmodification in advance, which not only involves tedious processes, butalso provides a modified fluorochrome with significantly decreasedquantum efficiency after dissolved in the aqueous solution, thus causingan adverse effect to sensitivity of the fluorescence detection.

In order to overcome the above disadvantages, the present inventorssurprisingly find that the method including encapsulating thefluorochrome selected from the group consisting of organic moleculesincluding those shown as formula (I), formula (II) or formula (III), orcarbon nanotubes, or PbS, PbSe or InAs quantum dots, or rare earthnanoparticles within the polymer microsphere by swelling achieves adesirable effect with high quantum efficiency of 25% or more and gooddispersibility in an aqueous solution, thereby facilitating labelingdetection of various biological macromolecule, with wide applicabilityfor different fluorochrome. Further, the present inventors also findthat when a binding region of a test strip is coated with an antibodylabeled with the near-infrared II fluorescent microsphere prepared bythe above method, the result detected by the test strip can be highlyaccurate, sensitive and precise

According to some embodiments of the present disclosure, provided are atest strip for short-wave near infrared immunofluorescencechromatographic detection, a system for immunofluorescencechromatographic detection and a method for quantifying an analyte in asample, which will be described in detail in the following description.

A Test Strip for Short-Wave Near Infrared ImmunofluorescenceChromatographic Detection

In one aspect of the present disclosure, provided is a test strip forshort-wave near infrared immunofluorescence chromatographic detection,by which an analyte in a sample can be detected accurately, sensitivelyand precisely. To facilitate understanding, the test strip forshort-wave near infrared immunofluorescence chromatographic detection isdescribed by reference to FIG. 1.

In embodiments of the present disclosure, the test strip for short-wavenear infrared immunofluorescence chromatographic detection includes:

a body 1, defining a sample region 100, a binding region 200, adetecting region 300 and an adsorbing region 400 connected with oneanother sequentially;

a first antibody, labeled with a fluorescent microsphere emitting awavelength in a range of 1000 nm to 1700 nm under an excitation lightless than 1000 nm, coated on the binding region 200 and configured tospecifically recognize an analyte;

a testing line 310 and a quality control line 320, located in thedetecting region 300, in which the testing line 310 closes to thebinding region 200;

a second antibody, coated on the testing line 310 and configured tospecifically recognize the analyte; and

a third antibody, coated on the quality control line 320 and configuredto specifically recognize the first antibody.

To facilitate understanding, the principle of detection by the presenttest strip for short-wave near infrared immunofluorescencechromatographic detection is described as follows.

The first antibody labeled with the fluorescent microspheres which iscoated on the binding region and the second antibody which is coated onthe testing line (refer to a T line) bind to two different sites in ananalyte (refer to an antigen) respectively. On the above basis, a samplecontaining an analyte moves toward the direction of the adsorbing regionunder capillary force after applied to the sample region and arrives atthe bonding region, at which the analyte is specifically bound to thefirst antibody firstly, thereby obtaining a first complex composed offluorescent microsphere-first antibody-antigen. Next, the first complexobtained continues to move toward the direction of the adsorbing regionunder capillary force and arrives at the testing line in the detectingregion, at which the first complex is specifically bound to the secondantibody, thereby obtaining a second complex composed of fluorescentmicrosphere-first antibody-antigen-second antibody in a sandwichstructure, in which the antigen contains two different sites bound bythe second antibody and the first antibody respectively. As the secondcomplex formed at the testing line accumulates over time, thefluorescence generated in the testing line is of an increased intensity.On the other hand, those first antibody labeled with the fluorescentmicrospheres which is not bound to the analyte also moves toward thedirection of the adsorbing region under capillary force and arrives atthe quality control line (refer to a C line), at which the firstantibody is specifically bound to the third antibody (a secondaryantibody specifically recognizing the first antibody) since there existsno interaction between the analyte and the third antibody, therebyobtaining a third complex between the first antibody and the thirdantibody. As the third complex formed at the quality control lineaccumulates over time, the fluorescence generated in the quality controlline is of an increased intensity till color development. A positiveresult is demonstrated by color development at both the testing line andthe quality control line. A negative result is demonstrated by weak ornone color development at the testing line but color development at thequality control line. None color development at the quality control lineindicates that the test trip for the immunofluorescence chromatographicdetection is invalid.

In embodiments of the present disclosure, the analyte is cardiactroponin, the first antibody is an antibody I against cardiac troponin(anti-CTNI1) (19C7, HyTest Ltd.), the second antibody is an antibody IIagainst cardiac troponin (anti-CTNI2) (16A11, HyTest Ltd.), and thethird antibody is a secondary antibody, preferably a goat-anti-mouseantibody, in which the first antibody recognizes the analyte at a firstsite different from a second site recognized by the second antibody.

In embodiments of the present disclosure, the fluorescent microspherelabeled to the first antibody is a near-infrared II polymer fluorescentmicrosphere prepared by the following steps:

1) dissolving a fluorochrome in a water-immiscible organic solvent, thusobtaining a fluorochrome solution; 2) distributing a polymer microsphereinto a sodium dodecyl sulfonate solution, thus obtaining a microspheresolution with the polymer microsphere as a carrier for the fluorochrome;3) subjecting a first mixture of the fluorochrome solution and themicrosphere solution to ultrasonic treatment, thus obtaining anemulsion; 4) swelling the emulsion such that the fluorochrome solutionenters nanopores formed during swelling of the polymer microsphere, thusobtaining a second mixture; and 5) heating the second mixture tovolatilize the organic solvent, such that the fluorochrome iscrystallized out and encapsulated in the nanopores, thus obtaining thenear-infrared II polymer fluorescent microsphere.

According to the method for preparing the near-infrared II polymerfluorescent microsphere in embodiments described above, the fluorochromeis dissolved in the organic solvent at first, thus obtaining thefluorochrome solution; meanwhile the polymer microsphere is distributedinto the sodium dodecyl sulfonate solution, thus obtaining a microspheresolution with the polymer microsphere as a carrier for the fluorochrome;subsequently the first mixture of the fluorochrome solution and themicrosphere solution is subjected to ultrasonic treatment, swelling andheating, such that the fluorochrome can be successfully encapsulated inthe microsphere, thus obtaining the near-infrared II polymer fluorescentmicrosphere.

According to the embodiments described above, in a simple and rapid way,the near-infrared II polymer fluorescent microsphere is prepared withgood dispersibility in an aqueous solution, high quantum efficiency upto 25%, and relative broader interval between an exciting wavelengthless than 1000 nm, such as at 365 nm or 740 nm, and an emittingwavelength at 1000 nm to 1700 nm. As a result, such a method not onlycan be widely used for various different fluorochrome, but also providesthe near-infrared II polymer fluorescent microsphere with strongpenetrability and low background interference during the fluorescencedetection, which can extremely magnify the fluorescent signal and thusimprove the sensitivity of the fluorescence detection, thereby havingpromising prospects in terms of live imaging, biolabeling detection andso on.

In embodiments of the present disclosure, the preparation of thenear-infrared II polymer fluorescent microsphere is described in detailwith reference to FIG. 2 and FIG. 3.

S100: Preparation of a Fluorochrome Solution

In embodiments of the present disclosure, a fluorochrome is dissolved ina water-immiscible organic solvent, thus obtaining a fluorochromesolution. In embodiments of the present disclosure, the fluorochrome isorganic molecules including those shown as formula (I), formula (II) orformula (III), or carbon nanotubes, or PbS, PbSe or InAs quantum dots,or rare earth nanoparticles

In embodiments of the present disclosure, the formula (I) is4,8-(5-(9,9-di(6-bromohexyl)-9H-fluoren-2-yl)-2,3-dihydrothieno[3,4-b][1,4]dioxine)-1H,5H-benzo[1,2-c:4,5-c′]bis([1,2,5]thiadiazole);the formula (II) is4,8-(5-(9,9-dihexyl-9H-fluoren-2-yl)-2,3-dihydrothieno[3,4-b][1,4]dioxine)-1H,5H-benzo[1,2-c:4,5-c′]bis([1,2,5]thiadiazole);and the formula (III) is2,3-dihydrothieno[3,4-b][1,4]dioxine)-1H,5H-benzo[1,2-c:4,5-c′]bis([1,2,5]thiadiazole).

In embodiments of the present disclosure, the fluorochrome selected fromthe group consisting of organic molecules including those shown asformula (I), formula (II) or formula (III), or carbon nanotubes, or PbS,PbSe or InAs quantum dots, or rare earth nanoparticles is used toprepare a near-infrared II polymer fluorescent microsphere. As shown inFIGS. 4 (a) to (c), absorption spectrums of and fluorescence spectrumsemitted respectively by fluorochrome of formula (I) to (III), theinventors find that the fluorochrome has strong absorption to lightsless than 1000 nm, such as at 365 nm and 740 nm, and emits fluorescencebetween 1000 nm and 1700 nm. Accordingly, because of the relativebroader interval between the exciting wavelength and the emittingwavelength, the fluorochrome has many advantageous characteristics suchas strong penetrability (penetration distance of several millimeters)and low background interference in the live imaging application, thuscan replace the conventional near-infrared fluorescent microsphere forapplication in imaging of living organisms, cells or tissues, diagnosingin vitro, and so on. Nevertheless, the fluorochrome as indicated abovecannot be directly applied in live imaging because of stronghydrophobicity per se, and thus requiring hydrophilic modification inadvance. The present inventors also find that the method includingencapsulating the fluorochrome as indicated above within the polymermicrosphere by swelling achieves a desirable effect with high quantumefficiency of 25% or more and good dispersibility in an aqueoussolution, thereby facilitating labeling detection of various biologicalmacromolecule. Therefore, the present near-infrared II polymerfluorescent microsphere prepared with the fluorochrome has advantageouscharacteristics such as strong penetrability and low backgroundinterference during the fluorescence detection, thereby having promisingprospects in terms of live imaging, biolabeling detection and so on.Further, the present inventors find that when such a near-infrared IIfluorescent microsphere prepared by the above method is applied inlabeling an antibody which is further coated on a binding region of atest strip, the result detected by the test strip can be highlyaccurate, sensitive and precise.

In a specific embodiment of the present disclosure, the fluorochrome inthe fluorochrome solution has a concentration between 1 mg/mL to 50mg/mL, specifically 1 mg/mL to 10 mg/mL, 10 mg/mL to 20 mg/mL, 20 mg/mLto 30 mg/mL, 30 mg/mL to 40 mg/mL or 40 mg/mL to 50 mg/mL, for example,1 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL or 50 mg/mL, preferably20 mg/mL, so that the polymer microsphere is capable of encapsulatingmore fluorochrome, and thus the present near-infrared II polymerfluorescent microsphere prepared emits the fluorescence with highintensity during the fluorescence detection, thereby further improvingsensitivity of the fluorescence detection.

In embodiments of the present disclosure, the organic solvent is atleast one selected from the group consisting of ethyl acetate,dichloromethane, trichloromethane, 1,2-dichloroethane and aromatichydrocarbons, in an example of the present disclosure, dichloromethane,so that dichloromethane can further facilitate swelling of the polymermicrosphere, thereby further increasing encapsulating efficiency of thefluorochrome.

S200: Preparation of a Microsphere Solution

In embodiments of the present disclosure, a polymer microsphere isdistributed into a sodium dodecyl sulfonate solution, thus obtaining themicrosphere solution with the polymer microsphere as a carrier for thefluorochrome.

In some embodiments of the present disclosure, the polymer microsphereis at least one selected from the group consisting of polystyrenemicrospheres, poly (methyl methacrylate) microspheres, polyformaldehydemicrospheres and poly (lactic acid-co-glycolic acid) microspheres. Sucha polymer microsphere is capable of encapsulating tens of thousands tohundreds of thousands of fluorescent molecules in one microsphere andpreventing the hydrophobic fluorochrome mentioned above from leaking outowing to a hydrophobic moiety inside the polymer microsphere; and iscapable of well dispersing in the aqueous solution due to a charge orhydrophilic moiety outside the polymer microsphere. Therefore, thenear-infrared II polymer fluorescent microsphere, with advantageouscharacteristics such as strong penetrability and low backgroundinterference during the fluorescence detection, can be prepared withsuch the polymer microsphere.

In embodiments of the present disclosure, the polymer microsphere has aparticle size of 20 nm to 1000 nm, specifically 20 nm to 100 nm, 100 nmto 200 nm, 200 nm to 300 nm, 300 nm to 400 nm, 400 nm to 500 nm, 500 nmto 600 nm, 600 nm to 700 nm, 700 nm to 800 nm, 800 nm to 900 nm or 900nm to 1000 nm, for example, 20 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm or 1000 nm, preferably 850nm. Accordingly, the polymer microsphere is capable of encapsulatingmore fluorochrome, such that the near-infrared II polymer fluorescentmicrosphere prepared can have high intensity of the fluorescence,thereby further improving sensitivity of fluorescence detection.

In embodiments of the present disclosure, the sodium dodecyl sulfonate(SDS) solution is of a concentration of 0.1% to 0.8%, for example 0.2%to 0.6%, eg., 0.25%, such that the SDS solution as an emulsifier notonly guarantees the polymer microspheres to be better dispersed in theSDS solution, but also facilitates obtaining a uniform emulsion duringthe subsequent ultrasonic treatment.

In embodiments of the present disclosure, the present polymermicrosphere is distributed into the sodium dodecyl sulfonate solution ina mass/volume ratio of 10 mg/mL to 200 mg/mL, specifically 10 mg/mL to50 mg/mL, 50 mg/mL to 100 mg/mL, 100 mg/mL to 150 mg/mL or 150 mg/mL to200 mg/mL, for example, 10 mg/mL, 30 mg/mL, 50 mg/mL, 70 mg/mL, 100mg/mL, 120 mg/mL, 150 mg/mL, 170 mg/mL or 200 mg/mL, preferably 30mg/mL, which not only enhances yield of the near-infrared II polymerfluorescent microsphere effectively, but also allows the polymermicrospheres to be swelled to a maximal extent in the next step, suchthat the polymer microsphere is capable of encapsulating morefluorochrome, and thus the present near-infrared II polymer fluorescentmicrosphere prepared emits the fluorescence with high intensity duringthe fluorescence detection, thereby further improving sensitivity offluorescence detection.

S300: Ultrasonic Treatment

In embodiments of the present disclosure, a first mixture of thefluorochrome solution and the microsphere solution is subjected toultrasonic treatment, thus obtaining an emulsion.

In embodiments of the present disclosure, the first mixture includes thefluorochrome solution and the microsphere solution in a volume ratio of1:5 to 1:20, specifically 1:5, 1:8, 1:10, 1:12, 1:15, 1:17 or 1:20,preferably 1:10, such that dichloromethane is in a proper amount forswelling all of the polymer microspheres thoroughly, and thefluorochrome solution is allowed to enter the nanopores formed duringswelling of the polymer microsphere completely, thus the polymermicrosphere is capable of encapsulating more fluorochrome and the highquality of near-infrared II polymer fluorescent microsphere prepared bythe present method emits the fluorescence with high intensity during thefluorescence detection, thereby further improving sensitivity offluorescence detection.

In embodiments of the present disclosure, the first mixture includes thefluorochrome and the polymer microsphere in a mass ratio of 0.1:100 to30:100, specifically 0.1:100, 0.5:100, 1:100, 5:100, 7:100, 10:100,12:100, 15:100, 20:100, 22:100, 25:100, 27:100 or 30:100, preferably1:15. Thus, the fluorochrome and the polymer microsphere are in such aproper matching ratio that availability of these raw material isimproved and the fluorochrome solution is allowed to enter the nanoporesformed during swelling of the polymer microsphere completely, thus thepolymer microsphere is capable of encapsulating more fluorochrome andthe near-infrared II polymer fluorescent microsphere prepared by thepresent method emits the fluorescence with high intensity during thefluorescence detection, thereby further improving sensitivity offluorescence detection.

S400: Swelling of the Emulsion

In embodiments of the present disclosure, the emulsion is swelled suchthat the fluorochrome solution enters nanopores formed during swellingof the polymer microsphere, thus obtaining a second mixture.

The present inventors find that a hydrophobic fluorochrome cannot bedirectly applied in live imaging, thus requiring hydrophilicmodification in advance, which not only involves tedious processes, butalso provides a modified fluorochrome with significantly decreasedquantum efficiency after dissolved in the aqueous solution. However, thepolymer microsphere used in the present disclosure is capable ofencapsulating tens of thousands to hundreds of thousands of fluorescentmolecules in one microsphere and preventing the hydrophobic fluorochromementioned above from leaking out owing to a hydrophobic moiety insidethe polymer microsphere; and is capable of well dispersing in theaqueous solution due to a charge or hydrophilic moiety outside thepolymer microsphere. Besides, such a polymer microsphere is allowed tobe modified from the outside with various functional groups in aflexible way, which facilitates labeling of molecules such as proteinsand DNAs. Accordingly, the present inventors provide the methodincluding encapsulating the fluorochrome selected from the groupconsisting of organic molecules including those shown as formula (I),formula (II) or formula (III), or carbon nanotubes, or PbS, PbSe or InAsquantum dots, or rare earth nanoparticles within the polymer microsphereby swelling to obtain the near-infrared II polymer fluorescentmicrosphere with high quantum efficiency of 25% or more and gooddispersibility in an aqueous solution, thereby facilitating labelingdetection of various biological macromolecules.

In embodiments of the present disclosure, the emulsion is swelled at 10°C. to 50° C. under stirring for 1 hour to 10 hours, specifically, at 10°C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C. or 50° C.,for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours or 10 hours, preferably, at 40° C. for 6 hours, such thatthe polymer microsphere can be swelled sufficiently in the presence ofdichloromethane, which ensures the fluorochrome entering nanoporesformed during swelling of the polymer microsphere successfully, and thusthe present near-infrared II polymer fluorescent microsphere preparedemits the fluorescence with high intensity during the fluorescencedetection.

S500: Volatilization of the Organic Solvent

In embodiments of the present disclosure, the second mixture is heatedto volatilize the organic solvent, such that the fluorochrome iscrystallized out and encapsulated in the nanopores, thus obtaining thenear-infrared II polymer fluorescent microsphere.

With slow volatilization of the organic solvent during heating, exteriorof the polymer microsphere is shrunk and the hydrophobic fluorochrome iscrystallized out (forming hydrophobic pellets), so that the fluorochromeis encapsulated inside the polymer microsphere. After the organicsolvent is totally volatilized, the near-infrared II polymer fluorescentmicrosphere is thus obtained, with the fluorochrome encapsulated insidebarely with leakage.

In embodiments of the present disclosure, the second mixture is heatedat a temperature of 50° C. to 90° C., specifically 50° C., 55° C., 60°C., 65° C., 70° C., 75° C., 80° C., 85° C. or 90° C., preferably 50° C.,so that the organic solvent, such as dichloromethane can be volatilizedquickly and completely in a short time period, so as to improve theefficiency of the present method.

In embodiments of the present disclosure, the second mixture is heatedunder magnetic stirring in a water bath at a temperature of 50° C. to90° C., so that the dichloromethane in the microsphere is volatilized ina high volatilizating rate, which improves the efficiency of the presentmethod.

In embodiments of the present disclosure, the method for preparing thenear-infrared II polymer fluorescent microsphere further includessubjecting the near-infrared II polymer fluorescent microsphere obtainedto ultrasonic cleaning with ethanol and water successively.

In embodiments of the present disclosure, after dissolved in a certainquantity in dichloromethane, the near-infrared II polymer fluorescentmicrosphere prepared by the present method is detected with itsfluorescence intensity based on a standard curve, with a calculatedresult of around 80,000 fluorescent molecules encapsulated in eachpolymer microsphere. Therefore, the near-infrared II polymer fluorescentmicrosphere prepared can emit a greatly magnified fluorescence signalwhen applied in labeling detection as compared with small moleculefluorochrome, thereby improving sensitivity of the fluorescencedetection.

A System for Immunofluorescence Chromatographic Detection

In embodiments of the present disclosure, the present system forimmunofluorescence chromatographic detection includes a fluorescenceimmunity analyzer, and a test strip for short-wave near infraredimmunofluorescence chromatographic detection described above, so that afluorescence signal can be generated in the test strip and then analyzedby the fluorescence immunity analyzer, thus achieving to qualitativelyor quantitatively detect an analyte, and improving accuracy, sensitivityand precision of the detected results as described above.

It should be understood by those skilled in the art that the featuresand advantages described above with respect to the test strip forshort-wave near infrared immunofluorescence chromatographic detectionare equally applicable to the system for immunofluorescencechromatographic detection, which will not be elaborated herein

A Method for Quantifying an Analyte in a Sample

In embodiments of the present disclosure, the method for quantifying theanalyte in the sample includes 1) applying the sample to a samplingregion of a test strip for short-wave near infrared immunofluorescencechromatographic detection described in the first aspect; 2) determininga fluorescence signal generated in the test strip of theimmunofluorescence chromatographic detection; and 3) quantifying theanalyte in the sample based on the fluorescence signal determined, thusimproving accuracy, sensitivity and precision of the detected results ina simple way.

In embodiments of the present disclosure, the sample is serum, thusfacilitating to sampling and obtaining a detection result with highaccuracy.

In embodiments of the present disclosure, the fluorescence signal isdetermined by a fluorescence immunity analyzer.

In embodiments of the present disclosure, the fluorescence immunityanalyzer is equipped with a color filter at a wavelength of 800 nm, thusbenefiting for obtaining fluorescence with high intensity, therebyfacilitating to observing and quantifying the fluorescence obtained.

In embodiments of the present disclosure, the method for quantifying theanalyte in the sample further includes determining fluorescence signalsin both a testing line and a quality control line in the step 2); andquantifying the analyte in the sample based on a ratio of thefluorescence signal generated in the testing line to the fluorescencesignal generated in the quality control line.

In embodiments of the present disclosure, the method for quantifying theanalyte in the sample further includes quantifying the analyte in thesample by means of a standard curve, based on the ratio of thefluorescence signal in the testing line to the fluorescence signalgenerated in the quality control line, in which the standard curve iscreated with serial cardiac troponin standards in concentrations of 50ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.2 ng/mL, 2.0 ng/mL, 1.0ng/mL, 0.5 ng/mL, 0.2 ng/mL and 0 ng/mL, thus contributing to improvingaccuracy of the detected result.

It should be understood by those skilled in the art that the featuresand advantages described above with respect to the test strip forshort-wave near infrared immunofluorescence chromatographic detectionare equally applicable to the method for quantifying the analyte in thesample, which will not be elaborated herein.

The embodiments of the present disclosure will be described in detail byreference to the following examples. It would be appreciated by thoseskilled in the art that the following examples are explanatory, andcannot be construed to limit the scope of the present disclosure. If thespecific technology or conditions are not specified in the examples, astep will be performed in accordance with the techniques or conditionsdescribed in the literature in the art or in accordance with the productinstructions. If the manufacturers of reagents or instruments are notspecified, the reagents or instruments may be commercially available.

Example 1

(1) Synthesis of a Carboxyl Polystyrene Microsphere

190 mL of water was added into a 500 mL round bottom flask and thenincubated in a water bath with a temperature of 70° C. under stirring ata speed of 350 rpm for half an hour. 16 mg of sodium dodecyl sulfate(SDS) as an emulsifier and 0.05 g of sodium bicarbonate as a bufferreagent were added and then incubated for another 10 minutes under thestirring. To the mixture, 8 mL of styrene and 0.8 mL of acrylic acidwere further added. After one hour, 0.2 g of potassium persulfate wasadded and an obtained reaction mixture was subjected to polymerizationreaction under nitrogen atmosphere for 18 hours. After completion of thereaction, the resulting product was centrifuged with a mixture ofethanol and water in a volume ratio of 2:1 (v/v) three times, thusobtaining the carboxyl polystyrene microsphere. The scanning electronmicroscopy of such a carboxyl polystyrene microsphere is shown in panelA of FIG. 5 (a). The carboxyl polystyrene microsphere was distributedinto a SDS solution at a concentration of 0.25% (w/v), thus obtaining a30 mg/mL carboxyl polystyrene microsphere dispersion as shown in FIG. 6(A) which was stored in a refrigerator under 4° C. for the next step.

(2) Synthesis of a Near-Infrared II Carboxyl Polystyrene FluorescentMicrosphere

40 mg of the near-infrared II fluorochrome represented by formula (I)was dissolved in 2 mL dichloromethane, thus obtaining a fluorochromesolution at a concentration of 20 mg/mL as shown in FIG. 6 (B). 20 mL ofthe carboxyl polystyrene microsphere dispersion obtained in (1) wasadded into a 500 mL conical flask and subjected to ultrasonic treatmentfor 5 minutes, after which 2 mL fluorochrome solution obtained above wasadded and subjected to ultrasonic treatment, thus obtaining an emulsion.Then the emulsion was subjected to magnetic stirring at a temperature of40° C. for 6 hours, such that the carboxyl polystyrene microsphereswelled sufficiently and the fluorochrome solution entered nanoporesformed during swelling of the polymer microsphere. The mixture swelledwas then subjected to magnetic stirring in a water bath at a temperatureof 50° C. overnight, so as to volatilize the dichloromethane in themixture completely. The product obtained was centrifuged, and thensubjected to ultrasonic cleaning with ethanol three times and with waterfor several times until the supernatant of the product centrifugedcontained no fluorochrome, thus obtaining the near-infrared II carboxylpolystyrene fluorescent microsphere which was distributed into water ina weight/volume of 5% and stored in a refrigerator under 4° C. for thenext step (FIG. 6 (C)).

(3) Evaluation of the Near-Infrared II Carboxyl Polystyrene FluorescentMicrosphere Obtained in (2)

3.1 In FIG. 6, FIG. 6 (A) shows that the solution of carboxylpolystyrene in SDS is white, FIG. 6 (B) shows that the solution offluorochrome represented by formula (I) in dichloromethane is cyan, andFIG. 6 (C) shows that the solution of carboxyl polystyrene fluorescentmicrosphere encapsulating the fluorochrome represented by formula (I) isalso cyan. Thus, it is demonstrated that the fluorochrome wassuccessfully encapsulated in the microsphere and properties on thesurface of the microsphere have not been changed significantly accordingto the color change. It also can be seen from the FIG. 6 (C) that thenear-infrared II carboxyl polystyrene fluorescent microsphere obtainedcan be distributed into water uniformly and stably.

3.2 Panel B of FIG. 5 (a) shows a scanning electron microscopephotograph of the near-infrared II carboxyl polystyrene fluorescentmicrosphere obtained in (2). It can be seen from FIG. 5 (a) thatmorphology of the carboxyl polystyrene microsphere has not been changedsignificantly before and after encapsulation of the fluorochrome, andthe fluorescent microspheres obtained are uniform in size and are notaggregated together.

3.3 Panel A of FIG. 7 (a) shows a fluorescent photograph of thecarboxylic polystyrene fluorescent microsphere obtained in (2) underirradiation with an excitation light at a wavelength of 740 nm, andpanel B of FIG. 7 (a) shows its fluorescent spectrum. It can be seenfrom the FIG. 7 (a) that the carboxylic polystyrene fluorescentmicrosphere can emit fluorescence in a wavelength between 800 nm and1700 nm nm with high fluorescence intensity, and its fluorescencequantum yield reaches 25% based on measurement.

(4) Application of the Near-Infrared II Carboxyl Polystyrene FluorescentMicrosphere Obtained in (2)

4.1 Coupling an Antibody to the Near-Infrared II Carboxyl PolystyreneFluorescent Microsphere

The near-infrared II carboxyl polystyrene fluorescent microsphereobtained in (2) was distributed into a 2-morpholinoethanesulfonic acid(MES) buffer (10 mM, pH 6.2), thus obtaining a uniform dispersion in aweight/volume ratio of 1%. To the dispersion,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) buffer (5 mg/mL) andsulfo-(N-hydroxysulfosuccinimide) (sulfo-NHS) buffer (5 mg/mL) wereadded for activation for 15 minutes. After centrifugation, thesupernatant was discarded and the remaining mixture was distributed intothe IVIES buffer (10 mM, pH 6.5) again. And then 0.4 mg/mL of MouseMonoclonal Antibody (anti-CTNI1 (19C7, HyTest Ltd.)) was added andincubated for 2 hours under stirring. The resulting mixture wascentrifuged again, the supernatant was discarded and the remainingpellets were distributed into 20 mM PBS containing 0.5% casein, 2.5%BSA, 1% sugar, 2% PEG-2000 and 0.03 wt % NaN₃ (pH 8.0) under ultrasonictreatment, thus obtaining a fluorescent microsphere dispersion coupledwith antibody anti-CTNI1 (1%, w/v) which was stored in a refrigeratorunder 4° C. for the next step.

4.2 Preparation of a Test Strip for Short-Wave Near InfraredImmunofluorescence Chromatographic Detection

The test strip for short-wave near infrared immunofluorescencechromatographic detection includes five parts, a supporting plasticboard, a sample pad, a binding pad, an adsorbing pad and anitrocellulose membrane, as shown in FIG. 8. Before assembled into thetest strip for short-wave near infrared immunofluorescencechromatographic detection completely, each part has to be pretreated.Specifically, the sample pad was soaked in a buffer containing 1% BSA,2% TritonX-100, 2% PVP 40, 20 mM Tris-HCl and 50 mM NaAc for 1 hour, andthen incubated at a temperature of 37° C. in an oven overnight; thefluorescent microsphere dispersion coupled with antibody anti-CTNI1obtained in the above step was sprayed uniformly onto the binding pad bya sprayer specialized for an immunofluorescence chromatographic test,subjected to lyophilization for 10 hours and stored for future use; and75 μL of anti-CTNI2 solution (1.0 mg/mL) (16A11, HyTest Ltd.) wassprayed uniformly onto a part of the nitrocellulose membrane by ascriber specialized for an immunofluorescence chromatographic detection,thus obtaining a testing line (that is a T line); in a similar way, 75μL of goat-anti-mouse antibody solution (0.5 mg/mL) (Hangzhou Qitaibiotechnology Co., LTD) was sprayed uniformly onto another part of thenitrocellulose membrane by the scriber, thus obtaining a quality controlline (that is a C line), after which the nitrocellulose membrane wasincubated at a temperature of 37° C. in an oven overnight. Then thesample pad, the binding pad, the nitrocellulose membrane and theadsorbing pad together were fixed neatly onto a hard cardboard along theaxis direction, in which the left margin of the adsorbing pad wasoverlapped with the right margin of the nitrocellulose membrane, theleft margin of the nitrocellulose membrane was overlapped with the rightmargin of the binding pad and the left margin of the binding pad wasoverlapped with the right margin of the sample pad, such that each partwas in close contact with its neighbor part, thus ensuring that thesample can move from the sample pad to the adsorbing pad smoothly.Finally, the cardboard assembled was cut into the test strips in a widthof 4 mm by a cutter for the test strip for short-wave near infraredimmunofluorescence chromatographic detection. The strip obtained wasthen packaged into an aluminium bag for storage.

4.3 Assembly of a System for Immunofluorescence ChromatographicDetection

The fluorescence signal generated in the test strip for short-wave nearinfrared immunofluorescence chromatographic detection was observed andanalyzed by a fluorescence immunity analyzer shown in FIG. 9 (A); and afluorescent spectrum of an analyte detected by the detection system isshown in FIG. 9 (B).

The light source of the fluorescence immunity analyzer is alight-emitting diode (LED) (365 nm, 1W); the divergent lights of LEDwere collimated by combined lenses; and Laser confocal optical systemwas used for irradiation and collection of fluorescence. When the teststrip for short-wave near infrared immunofluorescence chromatographicdetection applied with a sample was transported on a conveyor beltdriven by a stepper motor to expose under an excitation light,fluorescence was emitted at both the C line and the T line in thedetection region of the test strip. After passing through a color filterat a wavelength of 800 nm, the fluorescence emitted was focused to aphotosensitive panel of a silicon photocell sensitive to a light between500 nm to 1100 nm. An A/D (analog to digital) chip was configured totransform the voltage signal from the silicon photocell into a digitalsignal, thus obtaining a fluorescent spectrum of the sample. A lowermachine was configured to integrate peak areas corresponding to the Cline and the T line respectively, calculate the ratio of the peak areaof the C line to the peak area of the T line, and fit the concentrationsof standard samples, thus obtaining a standard curve which was saved inthe fluorescence immunity analyzer. During detection of a workingsample, the ratio of the peak area of the C line to the peak area of theT line was determined by the fluorescence immunity analyzer, and theconcentration of cardiac troponin in the working sample was quantifiedaccording to the standard curve.

4.4 Evaluation of the Test Strip for Short-Wave Near InfraredImmunofluorescence Chromatographic Detection

In order to preliminarily evaluate efficacy of the test strip or theimmunofluorescence chromatographic detection, cardiac troponin standardsdissolved in newborn calf serum at concentrations of 0 ng/mL and 20ng/mL were detected. Specifically, the cardiac troponin standards wereapplied to the sample region respectively; after 15 min, intensefluorescence was observed by a InGaAs camera at both the C line and theT line in the detection region of the test strip for short-wave nearinfrared immunofluorescence chromatographic detection under irradiationwith an excitation light at 808 nm (as shown in FIG. 10 (a)). For thecardiac troponin standard with a concentration of 0 ng/mL, very weakfluorescence was observed at the T line; while for the cardiac troponinstandard with a concentration of 20 ng/mL, intense fluorescence wasobserved at the T lime, indicating validity of the test strip or theimmunofluorescence chromatographic detection.strip forimmunofluorescence.

A standard curve of cardiac troponin was created as the following steps.Cardiac troponin standards in serial concentrations of 50 ng/mL, 25ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.2 ng/mL, 2.0 ng/mL, 1.0 ng/mL, 0.5ng/mL, 0.2 ng/mL and 0 ng/mL were formulated with newborn calf serum.Then 75 μL of the cardiac troponin standard at each concentration wasapplied to the sample region of the test strip and moved toward thedirection of the adsorbing region of the test strip under capillaryforce. After 15 minutes, the test strip was detected by the fluorescenceimmunity analyzer in triplicate, thus obtaining a mean value offluorescence intensity for each cardiac troponin standard. According,all mean values of fluorescence intensity obtained were curve-fittedwith corresponding concentrations of the cardiac troponin standards,with the standard curve generated and saved in the analyzer. Thefluorescent spectrums of the cardiac troponin standards were shown inFIG. 11, and the standard curve was shown in FIG. 13(a).

A serum sample was detected by the test strip for short-wave nearinfrared immunofluorescence chromatographic detection. 75 μL of a humanserum sample was applied to the sample region of the test strip andmoved toward the direction of the adsorbing region of the test stripunder capillary force. After 15 minutes, the test strip was detected bythe fluorescence immunity analyzer, and then the concentration of thecardiac troponin in the human serum sample was quantified base on thestandard curve created. The detection results obtained by the presenttest strip were compared with those from clinical detection, and thereexists consistency between them, as shown in FIG. 14 (a), thusdemonstrating the results detected by the present test strips areaccurate.

Example 2

(1) Synthesis of a Carboxyl Polystyrene Microsphere

All steps for synthesizing a carboxyl polystyrene microsphere are sameas those in Example 1. The scanning electron microscopy of the carboxylpolystyrene microsphere obtained is shown in panel A of FIG. 5 (b) and acarboxyl polystyrene microsphere dispersion obtained is same as thatshown in FIG. 6 (A).

(2) Synthesis of a Near-Infrared II Carboxyl Polystyrene FluorescentMicrosphere

All steps for synthesizing a near-infrared II carboxyl polystyrenefluorescent microsphere are similar with those in Example 1, except thatthe near-infrared II fluorochrome is represented by formula (II).

(3) Evaluation of the Near-Infrared II Carboxyl Polystyrene FluorescentMicrosphere Obtained in (2)

All steps for evaluating the near-infrared II carboxyl polystyrenefluorescent microsphere obtained are same as those in Example 1.

3.1 The solution of fluorochrome represented by formula (II) indichloromethane is also cyan, as shown in FIG. 6 (B); and the solutionof carboxyl polystyrene fluorescent microsphere encapsulating thefluorochrome represented by formula (II) is also cyan, as shown in FIG.6 (C). It also can be seen from the FIG. 6 (C) that the near-infrared IIcarboxyl polystyrene fluorescent microsphere obtained can be distributedinto water uniformly and stably.

3.2 Panel B of FIG. 5 (b) shows a scanning electron microscopephotograph of the near-infrared II carboxyl polystyrene fluorescentmicrosphere obtained in (2). It can be seen from FIG. 5 (b) thatmorphology of the carboxyl polystyrene microsphere has not been changedsignificantly before and after encapsulation of the fluorochromerepresented by formula (II), and the fluorescent microspheres obtainedare uniform in size and are not aggregated together.

3.3 A fluorescent photograph of the carboxylic polystyrene fluorescentmicrosphere obtained in (2) and its fluorescent spectrum are shown inFIG. 7 (b), which indicates that the carboxylic polystyrene fluorescentmicrosphere can emit fluorescence in a wavelength between 800 nm to 1700nm nm with high fluorescence intensity, and its fluorescence quantumyield reaches 25% based on measurement.

(4) Application of the Near-Infrared II Carboxyl Polystyrene FluorescentMicrosphere Obtained in (2)

4.1 Coupling an Antibody to the Near-Infrared II Carboxyl PolystyreneFluorescent Microsphere

All the steps for coupling an antibody to the near-infrared II carboxylpolystyrene fluorescent microsphere obtained in (2) are same as those inExample 1.

4.2 Preparation of a Test Strip for Short-Wave Near InfraredImmunofluorescence Chromatographic Detection

All the steps for preparing a test strip for short-wave near infraredimmunofluorescence chromatographic detection are same as those inExample 1.

4.3 Assembly of a System for Immunofluorescence ChromatographicDetection

All the steps for assembling a system for immunofluorescencechromatographic detection are same as those in Example 1.

4.4 Evaluation of the Test Strip for Short-Wave Near InfraredImmunofluorescence Chromatographic Detection

All the steps for evaluating the test strip for short-wave near infraredimmunofluorescence chromatographic detection are similar with those inExample 1, except that a standard curve of cardiac troponin was createdby using cardiac troponin standards in serial concentrations of 80ng/mL, 40 ng/mL, 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL,0.625 ng/mL, 0.3125 ng/mL and 0 ng/mL, and the fluorescent spectrums ofthe cardiac troponin standards were shown in FIG. 12, and the standardcurve was shown in FIG. 13 (b).

As shown in FIG. 10 (b), intense fluorescence was observed by a InGaAscamera at both the C line and the T line in the detection region of thetest strip for short-wave near infrared immunofluorescencechromatographic detection under irradiation with an excitation light at808 nm. For the cardiac troponin standard with a concentration of 0ng/mL, very weak fluorescence was observed at the T line; while for thecardiac troponin standard with a concentration of 20 ng/mL, intensefluorescence was observed at the T lime, indicating validity of the teststrip or the immunofluorescence chromatographic detection strip forimmunofluorescence.

As shown in FIG. 14 (b), there exists consistency between the detectionresults obtained by the present test strip and results obtained from aclinical detection, thus demonstrating the results detected by thepresent test strips are accurate.

Example 3

(1) Synthesis of a Carboxyl Polystyrene Microsphere

All steps for synthesizing a carboxyl polystyrene microsphere are sameas those in Example 1. The scanning electron microscopy of the carboxylpolystyrene microsphere obtained is shown in panel A of FIG. 5 (c) and acarboxyl polystyrene microsphere dispersion obtained is same as thatshown in FIG. 6 (A).

(2) Synthesis of a Near-Infrared II Carboxyl Polystyrene FluorescentMicrosphere

All steps for synthesizing a near-infrared II carboxyl polystyrenefluorescent microsphere are similar with those in Example 1, except thatthe near-infrared II fluorochrome is represented by formula (III).

(3) Evaluation of the Near-Infrared II Carboxyl Polystyrene FluorescentMicrosphere Obtained in (2)

All steps for evaluating the near-infrared II carboxyl polystyrenefluorescent microsphere obtained are same as those in Example 1.

3.1 The solution of fluorochrome represented by formula (III) indichloromethane is also cyan, as shown in FIG. 6 (B); and the solutionof carboxyl polystyrene fluorescent microsphere encapsulating thefluorochrome represented by formula (III) is also cyan, as shown in FIG.6 (C). It also can be seen from the FIG. 6 (C) that the near-infrared IIcarboxyl polystyrene fluorescent microsphere obtained can be distributedinto water uniformly and stably.

3.2 Panel B of FIG. 5 (c) shows a scanning electron microscopephotograph of the near-infrared II carboxyl polystyrene fluorescentmicrosphere obtained in (2). It can be seen from FIG. 5 (c) thatmorphology of the carboxyl polystyrene microsphere has not been changedsignificantly before and after encapsulation of the fluorochromerepresented by formula (III), and the fluorescent microspheres obtainedare uniform in size and are not aggregated together.

3.3 A fluorescent spectrum of the carboxylic polystyrene fluorescentmicrosphere obtained in (2) is shown in FIG. 7 (c), which indicates thatthe carboxylic polystyrene fluorescent microsphere can emit fluorescencein a wavelength between 800 nm to 1700 nm nm with high fluorescenceintensity, and its fluorescence quantum yield reaches 25% based onmeasurement.

(4) Application of the Near-Infrared II Carboxyl Polystyrene FluorescentMicrosphere Obtained in (2)

4.1 Coupling an Antibody to the Near-Infrared II Carboxyl PolystyreneFluorescent Microsphere

All the steps for coupling an antibody to the near-infrared II carboxylpolystyrene fluorescent microsphere obtained in (2) are same as those inExample 1.

4.2 Preparation of a Test Strip for Short-Wave Near InfraredImmunofluorescence Chromatographic Detection

All the steps for preparing a test strip for short-wave near infraredimmunofluorescence chromatographic detection are same as those inExample 1.

4.3 Assembly of a System for Immunofluorescence ChromatographicDetection

All the steps for assembling a system for immunofluorescencechromatographic detection are same as those in Example 1.

4.4 Evaluation of the Test Strip for Short-Wave Near InfraredImmunofluorescence Chromatographic Detection

All the steps for evaluating the test strip for short-wave near infraredimmunofluorescence chromatographic detection are same as those inExample 1, and the standard curve was shown in FIG. 13 (c).

As shown in FIG. 14 (c), there exists consistency between the detectionresults obtained by the present test strip and results obtained from aclinical detection, thus demonstrating the results detected by thepresent test strips are accurate.

Throughout this specification, reference to “an embodiment”, “someembodiments”, “one embodiment”, “another example”, “an example”, “aspecific example” or “some examples” means that a particular feature,structure, material or characteristic described in connection with theembodiment or example is included in at least one embodiment or exampleof the present disclosure. Thus, the appearances of the phrases such as“in some embodiments”, “in one embodiment”, “in an embodiment”, “inanother example”, “in an example”, “in a specific example” or “in someexamples” in various places throughout this specification are notnecessarily referring to the same embodiment or example of the presentdisclosure. Furthermore, the particular features, structures, materialsor characteristics may be combined in any suitable manner in one or moreembodiments or examples. In addition, it will be apparent to thoseskilled in the art that different embodiments or examples as well asfeatures of the different embodiments or examples described in thisspecification may be combined without contradictory.

Although explanatory embodiments have been shown and described, it wouldbe appreciated by those skilled in the art that the above embodimentscannot be construed to limit the present disclosure, and changes,alternatives and modifications can be made in the embodiments withoutdeparting from spirit, principles and scope of the present disclosure.

1. A test strip for short-wave near infrared immunofluorescencechromatographic detection, comprising: a body, defining a sample region,a binding region, a detecting region and an adsorbing region connectedwith one another sequentially; a first antibody, labeled with afluorescent microsphere emitting a wavelength in a range of 1000 nm to1700 nm under an excitation light less than 1000 nm, coated on thebinding region and configured to specifically recognize an analyte; adetecting line and a quality control line, located in the detectingregion, wherein the detecting line closes to the binding region; asecond antibody, coated on the detecting line and configured tospecifically recognize the analyte; and a third antibody, coated on thequality control line and configured to specifically recognize the firstantibody, wherein the fluorescent microsphere is a near-infrared IIpolymer fluorescent microsphere prepared by the following steps: 1)dissolving a fluorochrome in a water-immiscible organic solvent, thusobtaining a fluorochrome solution; 2) distributing a polymer microsphereinto a sodium dodecyl sulfonate solution, thus obtaining a microspheresolution with the polymer microsphere as a carrier for the fluorochrome;3) subjecting a first mixture of the fluorochrome solution and themicrosphere solution to ultrasonic treatment, thus obtaining anemulsion; 4) swelling the emulsion such that the fluorochrome solutionenters nanopores formed during swelling of the polymer microsphere, thusobtaining a second mixture; and 5) heating the second mixture tovolatilize the organic solvent, such that the fluorochrome iscrystallized out and encapsulated in the nanopores, thus obtaining thenear-infrared II polymer fluorescent microsphere.
 2. The test strip forshort-wave near infrared immunofluorescence chromatographic detectionaccording to claim 1, wherein the analyte is cardiac troponin, the firstantibody is an antibody I against cardiac troponin, the second antibodyis an antibody II against cardiac troponin, and the third antibody is asecondary antibody, preferably a goat-anti-mouse antibody, wherein thefirst antibody recognizes the analyte at a first site different from asecond site recognized by the second antibody.
 3. The test strip forshort-wave near infrared immunofluorescence chromatographic detectionaccording to claim 1, wherein the fluorochrome is organic moleculesincluding those shown as formula (I), formula (II) or formula (III), orcarbon nanotubes, or PbS, PbSe or InAs quantum dots, or rare earthnanoparticles


4. The test strip for short-wave near infrared immunofluorescencechromatographic detection according to claim 1, wherein the fluorochromein the fluorochrome solution has a concentration of 1 mg/mL to 50 mg/mL.5. The test strip for short-wave near infrared immunofluorescencechromatographic detection according to claim 1, wherein the organicsolvent is at least one selected from the group consisting of ethylacetate, dichloromethane, trichloromethane, 1,2-dichloroethane andaromatic hydrocarbons, preferably dichloromethane.
 6. The test strip forshort-wave near infrared immunofluorescence chromatographic detectionaccording to claim 1, wherein the polymer microsphere is at least oneselected from the group consisting of polystyrene microspheres, poly(methyl methacrylate) microspheres, polyformaldehyde microspheres andpoly (lactic acid-co-glycolic acid) microspheres.
 7. The test strip forshort-wave near infrared immunofluorescence chromatographic detectionaccording to claim 1, wherein the polymer microsphere has a particlesize of 20 nm to 1000 nm.
 8. The test strip for short-wave near infraredimmunofluorescence chromatographic detection according to claim 1,wherein in the step 2), the polymer microsphere is distributed into thesodium dodecyl sulfonate solution in a mass/volume ratio of 10 mg/mL to200 mg/mL.
 9. The test strip for short-wave near infraredimmunofluorescence chromatographic detection according to claim 1,wherein in the step 3), the first mixture comprises the fluorochromesolution and the microsphere solution in a volume ratio of 1:5 to 1:20.10. The test strip for short-wave near infrared immunofluorescencechromatographic detection according to claim 1, wherein in the step 3),the first mixture comprises the fluorochrome and the polymer microspherein a mass ratio of 0.1:100 to 30:100.
 11. The test strip for short-wavenear infrared immunofluorescence chromatographic detection according toclaim 1, wherein in the step 4), the emulsion is swelled at 10° C. to50° C. under stirring for 1 hour to 10 hours.
 12. The test strip forshort-wave near infrared immunofluorescence chromatographic detectionaccording to claim 1, wherein in the step 5), the second mixture isheated at a temperature of 50° C. to 90° C.
 13. A system forimmunofluorescence chromatographic detection, comprising: a fluorescenceimmunity analyzer; and a test strip for short-wave near infraredimmunofluorescence chromatographic detection according to claim
 1. 14. Amethod for quantifying an analyte in a sample, comprising: 1) applyingthe sample to a sampling region of a test strip for short-wave nearinfrared immunofluorescence chromatographic detection according to claim1; 2) determining a fluorescence signal generated in the test strip forthe immunofluorescence chromatographic detection; and 3) quantifying theanalyte in the sample based on the fluorescence signal determined. 15.The method according to claim 14, wherein the sample is serum.
 16. Themethod according to claim 14, wherein the fluorescence signal isdetermined by a fluorescence immunity analyzer.
 17. The method accordingto claim 16, wherein the fluorescence immunity analyzer is equipped witha color filter at a wavelength of 800 nm.
 18. The method according toclaim 14, further comprising: determining fluorescence signals in both adetecting line and a quality control line in the step 2); andquantifying the analyte in the sample based on a ratio of thefluorescence signal generated in the detecting line to the fluorescencesignal generated in the quality control line.
 19. The method accordingto claim 18, further comprising: quantifying the analyte in the sampleby means of a standard curve, based on the ratio of the fluorescencesignal generated in the detecting line to the fluorescence signalgenerated in the quality control line, wherein the standard curve iscreated with serial cardiac troponin standards in concentrations of 50ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.2 ng/mL, 2.0 ng/mL, 1.0ng/mL, 0.5 ng/mL, 0.2 ng/mL and 0 ng/mL.